Today, antibodies are widely used agents in the field of medicine and research. In medicine, they find application in many different fields. For example, antibodies are used as labeling agents for detecting certain markers which allow the diagnosis and/or prognosis of diseases or the determination of specific body parameters such as, for example, the presence or concentration of certain hormones.
Furthermore, antibodies are also used as therapeutic agents in the treatment and prophylaxis of a variety of diseases such as cancer, cardiovascular diseases, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infections. In these therapies, the antibody may possess therapeutic activity on it own, for example by blocking receptors or messenger molecules, thereby inhibiting their disease-relevant functions, or by recruiting and activating components of the patient's immune system. Alternatively, the antibody may be coupled to another agent having therapeutic activity. In particular in the treatment of cancer and infections, said further agent has cell-killing activity and may be, for example a radioisotope or a cytotoxin. In another application, antibodies may be used to passively immunize a patient by transferring suitable antibodies into the patient's circulation.
Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat, rabbit, goat, sheep, or horse. Blood isolated from these animals contains polyclonal antibodies directed against said antigen in the serum. To obtain an antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line, resulting in hybridoma cells. Single hybridoma cells are then isolated by dilution cloning to generate cell clones that all produce the same monoclonal antibody.
However, in therapeutic applications these monoclonal antibodies have the problem that they are derived from animal organisms and differ in their amino acid sequence from human antibodies. The human immune system hence recognizes these animal antibodies as foreign and rapidly removes them from circulation. Furthermore, systemic inflammatory effects may be caused. A solution to this problem is the replacement of certain constant parts of the monoclonal antibody with corresponding parts of a human antibody. If only the heavy and light chain constant regions are replaced, a chimeric antibody is obtained, while the additional replacement of the framework regions of the heavy and light chain variable regions results in so called humanized antibodies.
In research, purified antibodies are used in many applications. They are most commonly used to identify and locate biological molecules such as in particular proteins. The biological molecules may either be detected after they have been isolated, for example to determine their presence, concentration, integrity or size. On the other hand, they may be detected in cellular or tissue samples, for example to determine their presence or location. Furthermore, antibodies are used in isolation procedures of specific biological substances, in particular proteins, wherein the antibody specifically separates the biological substance of interest from the sample containing it.
In all these applications, a tight binding and specific recognition of the antigen is of vital importance for the antibody used. Thereby, higher activity and less cross-reactivity, in particular less adverse side effects in therapeutic applications, are obtained. However, during humanization of monoclonal antibodies, often the affinity and specificity of the engineered antibody is decreased.
An interesting and important group of antibodies are those directed against mucin proteins. Mucins are a family of high molecular weight, heavily glycosylated proteins produced by many epithelial tissues in vertebrates. They can be subdivided into mucin proteins which are membrane-bound due to the presence of a hydrophobic membrane-spanning domain that favors retention in the plasma membrane, and mucins which are secreted onto mucosal surfaces or secreted to become a component of saliva. The human mucin protein family consists of at least the family members MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, and MUC20; wherein MUC1, MUC3A (isoform 1), MUC3B and MUC4 are membrane bound.
Increased mucin production occurs in many adenocarcinomas, including cancer of the pancreas, lung, breast, ovary, colon, etc. Mucins are also overexpressed in lung diseases such as asthma, bronchitis, chronic obstructive pulmonary disease or cystic fibrosis. Two membrane mucins, MUC1 and MUC4 have been extensively studied in relation to their pathological implication in the disease process. Moreover, mucins are also being investigated for their potential as diagnostic markers.
Several antibodies directed against mucin proteins, in particular MUC1, are known in the art. Some of them are already approved for medical applications. However, their use could still be improved if their antigen affinity and/or specificity could be enhanced.
In view of this, there is a need in the art to provide improved anti-mucin antibodies preferably having enhanced antigen binding and/or recognition properties as well as methods which are suitable to improve the antigen binding and/or recognition of known antibodies, in particular of therapeutic MUC1 antibodies.